Process for producing butyl alcohol and acetone by fermentation



Patented July 11, 1933 HUGO W'EBTHEIM, OF VIENNA, AUSTRIA :enocnss non rnonuctnve BUTYL ALcoHoL AND aonronn BY'FERMENTATIQN Ho Drawing. Application filed February 13, 1930, Seria1 170.42%,235, and inAustria February 23, 1929.

the United States of America until it attained.

great industrial importance. Both amylaseous and saccharine natural crude materials may be employedas starting products, since the organisms setting up bacterial butylacetonic fermentation are capable. of easily degrading starch-to monosaccharides by enzymatic action. Butyl alcohol and acetone havebeen formed in the fixed proportion of 2 to 1 from all organisms hitherto employed. The yield of solvents varies between 21% and 25% calculated onthe dry mash. In addition thereto carbon-dioxide and hydrogen 25 are obtained as gaseous ucts.

The development of this method of fermentation up to the state of the industry at the present time has been facilitated particularly by the discovery of the fact that in order to obtain uniformly vigorous fermentation,

fermentation prodcultures must be employed at the start, which,

contain no vegetative forms of growth but consist excluslvely of spores. Cultures are accordingly employed for inoculating purposes which have been heated to about 95 C;

for a short time. It has moreover transpired that the acids produced by fermentation must not be neutralized with chalk. Onthecontrary in order that the'fermentation should proceed along the correct lines it is essential that the acidity should from the start continuously increase until it reaches a maximum (peak) and should thereafter decrease again continually until the completion of the fermentation. The fermentation curve constitutes an important method of controlling the operation; when the acidity decreases or thatthe organism setting up fermentation itself is attenuated. The carbohydrate. content of the mashes must be considerably below the concentrations which are permissible U for alcoholic fermentation; the upper limit very slowly or does not even decrease at all,- this 18 a sure s gn that the meshes are infected is defined by 8% starch. However, the viscosity of the mashes which are produced by steaming the starting materials under pressure, especially when maize meal is employed, is considerable, notwithstanding lower concentrations, and it has thereforebeen proposed to carry out this boilingunder pressure with the addition ofsuch limited amount of hydrochloric acid as is just sufficient to convert the diphosphates containedin the flour into 'monophosphates. Finally Fernbach has already recommended to add where necessary to the carbohydrate mash decomposed or partially degradedyeast to act as'a nutritive substance. In addition to thesemore or less important proposals for the technical development of theprocess, there are to be found more particularly in patent specifications, data concerningvarious organisms to which specific properties have been ascribed suiting them for this process.

According to the present invention the main fermentation is allowed to proceed in mashes which are acidulated prior to the initiation of the fermentation by theaddition of organic acids, more particularly' lactic acid. No matter what particular organism suitable for carrying out the butyl-acetonic fermentation is employed, the prevention of contamination and normal course of the fermentationis by this means assured. is known, organic acids act in admixture with. their own salts (formed with strong bases), as buffers and thus prevent the hydrogen ion ooncentration from exceeding certain limits .in spite of the increase'of the acidity during the fermentation. This special action which is possessed by organic acidatOge-ther with the power of suppressingthe danger of con taminated fermentation setting in, cannot be obtained by the addition of inorganic acids. The self regulating butler action of the sys-.' tem, organic acids-salts of these acids With strong bases, is with advantage still further increased by the addition of killed or autolyzed yeast (known per 'se) or by the-addi tion;.of other mixtures of substances with good buffer properties, which'iat the same time form nitrogeneous nutritive substances, such as albuminaceous mixtures of vegetable origin. In addition the admixture of ammonium compounds 'is recommended, particularly of ammonium salts or of urea or the like. v

Instead of adding. organic salts to the mash they can be produced in the mash by fermentation, thus for example, lactic acid may be produced'hy the lactic acid fermentation customaryin the production of yeast.

The initial acidity of the mash (titrable acidity towards bromthymol blue) is preterably adjusted so as to amount to from 1.4 to 1.6 c. c. normal NaOH per 100 c. c. The pH value at the beginning of the fermentation should lie between 5 to 4.6, the degree 11' to which the buffering is carried being at least 4. V

The buffering index 7r (to which Michaelis, Hydrogen Ion Concentration, 1926 edition, page 106, assigns the symbol is expressed mathematically by the equation The numerical value of the buffering is, as is known, determined by measuring the variations of the pH Value occurring with the addition of a delinitesmall amount of acid or'alkali to a. given amount of the culture medium. Theoretically, the additions should be infinitely small, but in practice the lower limit is set'by the accuracy of the method of pH determination. In order to obtain comparable results, the amount of added acid or alkali must be constant in all the experiments. I he 71' values given below are based on the following known-method of determination. Three test portions of the culture medium, each about 1 c. c., are taken out together. In one of these test portions the pH value is measured by the usual method. To the second test portion 0.5 c. c. of

sulphuric acid'solution, to the third 0.5 c. c. r

{II/100 solution of caustic soda are added, whereupon the pH value is also determined 'inthese two test portions by the same method as above. The changesin the pH value are indirectly proportional to the buffering value 1r. a

Theoretically, the amount of these alterationsshould be of equal value, with opposite signs, but in practice, there are often considerable discrepancies between the changes of the pH value, which are produced by equivalent amounts ofacids and akali's. In the practical determination after having fixed the changes of pH towards the acid and alkaline sides the arithmetical mean of the reciprocal values is calculated and. accepted as the. value 'of 11'. For determining the pH value the known indicatoustripmethod of Dr Peter Wulff (see German Patent No. 405.691)wasemployed. I

It is recommended to supervise the course of the fermentation by'periodically measui'- ing the buffer index w employing this method of determination ;1 n order to adapt the ir value to the change in the acidity so that said value increases with the acidity and preferably attains its maximum simultaneously with the maximum acidity. If the buffer index is found to be insuiiicicntly high during the course of the main fermentation, then E mamp Ze 100 litres of the mash are to contain about -37 kilos of potatoes or 9-10 kilos of maize meal. Other starchy materials are worked up into 'mashes of the same. concentration (67% of pure starch) according to their content of starch. The potatoes are used in the whole form, the maize of the like if the form of meal, being introduced with a suitable quantity of water into an autoclave which is provided with agitating means, where they are steamed for about 1-2 hours under a pressure of 2-3 atmospheres. The nitrogenous nutrient substances serving at the same time as buiier (killed or autolyzed yeast, or malt sprouts in conjunction with urea or ammonium phosphate or ammonium sulphate, or similar nitrogenous in various combinations) are preferably add ed to'the' mash while in the autoclave. A typical suitable composition is for example per 100 litres of mash, 125 grams of killed or degraded yeast, 125 gramsof malt sprouts and 40 grams of ammonium sulphate. The mash is blown directly into the fermentation tank which has been previously carefully sterilized and'which is provided with agitating-means, said tank being completely closed and provided with a top adaptedto collect the liberated gr After adjusting the suitable initial acidity to 1.4 te 1.6 c. c. normal NaOH per 1000. c. for which purpose -90 c. c. of 80% lactic acid are preferably added to every litres of the mash, the latter is then reduced to the fermentation temperature of '37'38, for example, by cooling devices internally disposed in the vessel; however for this purpose the mash can also be caused to pass through heat-exchange apparatus. The mash shows new a pH'value between 5 and-1.6 and a butler-index of at least at.

Whenthe mash is ready for pitching a suitable quantity of the bacterial pitching material must also be ready fornse. For this purpose for example a'sterile carbohydrate mash is prepared somewhat inthe same manner as the main mash and cc. of this culture medium are inoculated with a spore culture of the species B. ctmg lobacter A. ill. and Bredemmm (named Cllostrz'alc'mnv butym'cum Praemo'wsicflby the Society of American Bacteriologists) which culture has been provisionally empirically tested as to its capacity of producing butyl-aceto-nic fermentation, and this'culture is left to developat 3738 C. under anaerobic conditions. The growth begins after. about 48 hours and therewith the development of the oidium. After it has been ascertained that the culture is of unimpeachable character, it is used to inoculate about 2 litres ofa similar sterilized mash and after about 24 hours is transferred to about 20 litres of a similar mash. After a further 24 hours have elapsed these 20 litres are introduced into a prepared 'mash having a volume of 200 to 300'litres which after a further 24 hours is transferred to the main mash which has been prepared-in the meantime.

During the fermentation of the main mash in addition to the bacteriological examination and the control-of the titrable acidity, the

pH value and the buffer concentration are determined at least four times daily which leads under certain circumstances to the ,addition of further buffer substances. After fermentation has proceeded for about 30 to 32 hours, during whichthe fermenting mixture must be carefully stirred at intervals, the culminating point of the fermentation and therewith the maximulm acidity. (peak) is reached.

After the fermentation is terminated the main products formed (butyl alcohol and acetone) are recovered'as is customary by fractional distillation. The presence of small amounts of other alcohols does not render a separate recovery thereof economical.

The gases evolved during the fermentation, i. e. hydrogen and carbon dioxide, as well as the residues containing valuable feeding stuffs, can be utilized in the customary manner.

In the present process thereis no need of using micro-organisms immunized in any way to the presence of acids, in spite of the fact that bacteria of the species Amylobacter A. 31. and Brcdemamt which perferably have to be used with the presentprocess, areeX- tremely sensitive to acids. As is well known, preferably for each large scale fermentation a setting onportion is prepared, starting each time from spores of a laboratory culture. Consequently each culture is passed only once through the culture medium acidulated before initiating fermentation and may stand this without suffering much dam- Nevertheless butyl-acetomc bacilli immuthe fermented mash;

nized to acid may be used with advantage.- A process for carrying out the fermentation by means of bacteria of the species Amy/Z0- ba cter A. M. and Bredemmm which have been habituated, by cultivation, to increasing amounts ofacids, and a special method for cultivating such organisms particularly adapted for the process described and claimed in the present application, constitutes the subject matter of my copending application Serial No. 428,234 filed February 13, 1930. This process consists essentially in continuing to cultivate bacteria of the species Amylobacter A. ill. and Breclemcmn 011 nutrient media with progressively increasing initial acidity, and without neutralizing the acid produced by the fermentation, until the organism has become acidproof to the desired extent, whilst between each two successive cultivations on such a nutrient medium there is interposed a fermentation in a neutral or alkaline medium, in -which fer-' mentation the acid formed is neutralized. The cultures have to be heated for a short time, in the usual way, prior to inoculation of the nexthalftuating fermentation with higher initial acidity, in order to kill all the vegetative forms so that spores alone are subculti'vated-in the habituating fermentation. The degree of acidity indicated in the following cla ms are based ona determination using bromthymol blue as indicator.

I claim: I

1. A process for the production of butyl alcohol and acetone by fermentation which compri es preparing a sterile carbohydrate mash, acidulating it. beforelin itiating fermentation, inoculating said mash with bacteriaof the species Amylobacter A. M. and Bredcmami, hutyl-acetonic bacilli, permitting the fermentation to go to completion and recovering butyl alcohol and acetone from 2. In a'process as setferth in claim 1, the step consisting in that the titratable init al acidity determined with bromthymol blue as indicator is adjusted to 1.4- to1.6 c. 0. normal NaOH per 100 cc.

3. A'process for the production of butyl alcohol and acetone by fermentation which comprises preparing a sterile carbohydrate mash, adding buffering mixtures thereto, ad-

justing the pH value to 5 to 1.6 for a buffer index 7.- of at least 4,'inoculating said mash with bacteria of the species Amylobaczfcr A. M. and Emblems mt, butyl-acetonic bacilli, permitting the fermentation .to go to com- ,pletion and recovering butyl alcohol and acetone from the fermented mash.

4. A process for the production of butyl alcohol and acetone by fermentation which comprises preparing a sterile carbohydrate mash acidulating the said mash-up to an acidity of at least mere 1.6 c. c. normal NaOH'per .100--c.c., adding bulferingmixturesthereto to adjust the pH to a value between 5 and4a6 for a buffer index of at least 4, inoculating said mash with bacteria of the species Amylobacter A. M. and Brcdemaom, butyl-acetonic bacilli, permitting the fermentation to go to completion and recovering butyl alcohol and acetone from the fermented mash.

5. A process for the production of butyl alcohol and acetone by fermentation, which comprises preparing a. sterile carbohydrate mash, adjustng the hydrogen ion concentration of the said mash, by addition of a non-volatile organic acid, to a pH of less than 5, inoculating the said mash with butylacetonic bacteria of the species Amylobactcr A. 171 and B'iGdG'DUUIl IL, permitting the fermentation to go to completionand recovering butyl alcohol and acetone from the fermented mash. I

. 6. A process for the manufacture of butyl alcohol and acetone by fermentation which comprises preparing a sterile carbohydrate mash, adjusting the hydrogen ion concentration of the said mash, by addition of an organic acid, to a pH of less than 5, inoculating said mash with bacteria of the species .Jl YH I/ZObQCZ (W' A. M. (and B'ICflibfiht'flll, and fer.- menting the said mash to produce mainly butyl alcohol and acetone.

7. A process for the manufacture of butyl alcohol and acetone by fermentation which comprises preparing a: sterile carbohydrate mash, adjusting the hydrogen ion concentration of the said mash, by addition of lactic acid, to a pH of less than 5, inoculatingsaid mash with bacteria of thespecies Amy Z0- ?)(zcter A. ill. and Brahma-1m, and fermenting the said mash to produce mainly butyl alcohol and acetone.

8. A process for the manufacture of butyl alcohol and acetone by fermentation which comprises preparing a sterile carbohydrate mash, adjusting the hydrogen ion concentration of the said mash, by fermentative generation of lactic acid, to a pH ofless than 5, I

inoculating said mashwith' bacteria of the species A-my lobaoter A. M. and Bredemami, and fermenting the said mash to produce mainly butyl alcohol and acetone.

9. Process for the manufacture of butyl alcohol and acetone by fermentation which comprises preparing a sterile carbohydrate mash, acidulating it before initiating fer-men: tation, adding buffering mixtures thereto, inoculating said mash with but'yl-acetonic bacilli immunized, by previous cultivation, to the presence of acids, modifying in the course of the fermentation the buffer index, by furher addition of buffering substances, so as to suit the changes in titratableracidity, permittingthe fermentation to go to completion step which consists in modifying the pH value in the course of the fermentation to suitthe variation in the titratable acidity in such a way as to attain its highest value with the maximum acidity.

11. A process for the manufacture of butyl alcohol and acetone by fermentation which comprises preparing. a sterile carbohydrate masl1,,adjusting the hydrogen ion concentration of tlie'said mash, by addition of an acid, to a pH of less than 5, inoculating said mash with bacteria of the species Amylobactc-r A. M. and Brcdema-vm, and fermenting the said mash to produce mainly butyl alcohol and acetone.

12. Process for themanufacture of butyl alcohol and acetone by fermentation which comprises preparing a sterile carbohydrate mash, acidulatin g it before initiating fermentation, inoculating said mash with butyl-acetonic bacteria immunized, by previous cultivation, tothe presence of acids, and fermenting the said mash to produce mainly butyl alcohol 7 and acetone.

18. Process for the manufacture of butyl alcohol and acetone by fermentation which comprises preparing a sterile carbohydrate mash, adjusting the hydrogen ion concentration of the said mash,'by addition of an acid, to a pH of less than 5, adding buffering mixtures thereto, inoculating said mash with butyl-acetonic bacilli immunized, by previous cultivation, to the presence of acids, modifying during fermentation the buifer index, by further addition of buffering substances, so as to suit the changes in the titratable acidity, permitting the fermentationto go to comple tion and recovering butyl alcohol and acetone from the fermented mash. A

' 14-. Process for the manufacture of butyl alcohol and acetone by fermentation which comprises preparing a sterile carbohydrate mash, adjusting the hydrogen ion concentration of the said mash,by addition of an acid, to a pH of less than 5, adding buffering mixtures thereto which'serve at the same time as nitrogenous nutriment, inoculating said mashwith .butyl-acetonic bacilii immunized, by previous cultivation, to the presence of acids, modifying during fermentation the buffer index, by further addition of buffering substances, so as to suit the changes in the titratable acidity, permitting he fermentation togo to completion and recovering butyl alcohol and acetone from the fermented mash.

. In testimony whereof I have afiixed my signature.

HUGO WERTHEIM.

and recovering butyl alcohol and acetone from the fermented mash.

'10. In a process as claimed in claim 9 the 

